The cytochrome b6f complex has many structure-function homologies with the cytochrome bc1 complex of the mitochondrial respiratory chain. Much of this arises from the identity of cytochrome b, especially marked on the p-side of the membrane. From the high resolution structures of cytochrome f and the Rieske (ISP) protein, it is known that the peripheral domains differ significantly between bc1 and b6f. The further the domain from the membrane, the greater the difference. Thus, cytochromes f and c1 are completely different proteins and have been from their evolutionary origin, and the domain of the ISP distal to membrane folds differently in the b6f and bc1 complexes. However, the ISP domain containing the iron-sulfur cluster that must come close to the membrane has a conserved fold. Thus, detailed information on structure-function for the b6f complex complements that obtained for the bc1 and seems likely to provide differences in detailed functional mechanisms. A high resolution structure of 3-D crystals of the b6f complex from the thermophilic cy- anobacterium, M. laminosus, would provide this information. The crystals presently show ordered diffraction to 10 Angstrom units. When diffraction (less than or equal to 3 Angstrom units) appropriate for a structure analysis is obtained, the solution of the structure will be expedited by the fact that we have high resolution structures (less than 2.0 Angstrom units) for the p- side of the complex, cytochrome f and the iron-sulfur protein, 40 percent of the total mass of the complex. Issues of function to be determined by the structure include the position and function of the n-side quinone, the pathway of trans-membrane H+ transfer, and the role of intramembrane bound water. From the existing p- side structures, the local mobility of the ISP will be analyzed in vivo, in situ, and in vitro. The basis for the non-concerted reduction of high and low potential chains will be studied. Catalysis of electron transfer by conserved aromats in the Rieske protein, and in cytf where they shield the heme, will be tested my mutagenesis. The role of the water chain in the coupling of intraprotein electron and proton transfer will be examined by stopped flow kinetics in D2O, together with the properties of the bound H2O in cytf by FTIR. With a ruthenium derivative of cytf, "photo-cytf", light-induced intraprotein electron transfer rates, optimum paths of intraprotein electron transfer, and reorganization energy will be measured. Ruthenated cytf will also be used to investigate intraprotein protonation- deprotonation at specific carboxylates, associated with coupled electron and proton transfer.